I propose to study the enzymatic and molecular mechanisms of genetic recombination in E. coli. Several of the pathways for genetic recombination in E. coli have been shown to mediate the interconversion of certain plasmid DNAs and their circular oligomeric forms. Assays that measure the interconversion of plasmid DNAs and their circular oligomeric forms in vitro have been developed. The E. coli recA protein and recF protein will be purified by an in vitro complementation assay which measures the ability of these proteins to complement the effect of the recA and recF mutations on plasmid DNA oligomer metabolism in vitro. The recA protein, the recB, recC protein (exonuclease V) the recF protein and recE protein (exonuclease VIII) will be used in reconstitution assays to purify unknown proteins that are involved in the recB, recC, the recF and possibly the recE pathways of genetic recombination. These recombination pathways will be reconstituted from purified proteins and used to study the mechanisms by which plasmid DNAs recombine in vitro. DNA sequences which promote high levels of plasmid DNA circular oligomer formation will be isolated and the interaction of these DNA sequences with purified recombination proteins will be studied. An assay that measures the recombination of amber mutations in bacteriophage phix174 RF DNA monomers in vitro has also been proposed and will be used to study the exchange of genetic markers by purified proteins in vitro.